Conventional phosphoramidite chemistry, so named for a functional group on the monomer building blocks, was first developed in the early 1980's as disclosed in U.S. Pat. No. 4,415,732. This functional group provided a relatively efficient means of joining a building block monomer to the growing chain. Solid phase synthesis disclosed by Caruthers et al. in U.S. Pat. No. 4,458,066 was another improvement to oligonucleotide synthesis. In this technique, the growing DNA chain is attached to an insoluble support via a long organic linker which allows the growing DNA chain to be solubilized in the solvent in which the support is placed. The solubilized, yet immobilized, DNA chain is thereby allowed to react with reagents in the surrounding solvent and allows for the easy washing away of the reagents from the solid support to which the oligonucleotide is attached. These significant advances in phosphoramidite chemistry and solid phase synthesis paved the way to making custom DNA synthesis accessible to the average biology lab. Other novel techniques, e.g. polymerase chain reaction (“PCR”, which is used in forensic testing and DNA fingerprinting), have been facilitated due to the ready availability of synthetic DNA.
There are several sites on the nucleosides of similar chemical nature, e.g.——OH or hydroxyl groups. However, during oligonucleotide synthesis, the monomer subunits must be attached to the growing oligonucleotide molecule in a site-specific manner. This requires functionalizing a site either on the growing chain or on the incoming base for attachment of the incoming monomer building block to the growing chain. To prevent the incoming monomer from attaching at the wrong site, the wrong sites must be blocked while the correct site is left open to react. This requires the use of what are termed protecting groups. Protecting groups are compounds attached temporarily to a potentially reactive site so as to prevent it from reacting. The protecting group must be stable during said reactions and yet must eventually be removed to yield the original site. The synthesis of oligonucleotides requires several sites to be protected and particular sites must be deprotected while others remain protected. These protecting groups grouped together as a set are termed orthogonal protecting groups.
Solid phase oligonucleotide synthesis protocols typically use a dimethoxytrityl protecting group for the 5′ hydroxyl of nucleosides. A phosphoramidite functionality is utilized at the 3′ hydroxyl position. The synthesis generally proceeds from the 3′ to the 5′ of the ribose or deoxyribose sugar component of the phosphoramidite nucleoside in a synthesis cycle which adds one nucleotide at a time to the growing oligonucleotide chain. Beaucage et al. (1981) Tetrahedron Lett. 22:1859. See FIG. 1 for a schematic representation of this technology. In FIG. 1 “B” represents a purine or pyrimidine base, “DMT” represents dimethoxytrityl protecting group and “iPr” represents isopropyl. In the first step of the synthesis cycle, the “coupling” step, the 5′ end of the growing chain is coupled with the 3′ phosphoramidite of the incoming monomer to form a phosphite triester intermediate (the 5′ hydroxyl of the added monomer has a protecting group so only one new monomer is added to the growing chain per cycle). Matteucci et al. (1981) J. Am. Chem. Soc. 103:3185. Next, an optional “capping reaction” is used to stop the synthesis on any chains having an unreacted 5′ hydroxyl, which would be one nucleotide short at the end of synthesis. The phosphite triester intermediate is subjected to oxidation (the “oxidation” step) after each coupling reaction to yield a more stable phosphotriester intermediate. Without oxidation, the unstable phosphite triester linkage would cleave under the acidic conditions of subsequent synthesis steps. Letsinger et al. (1976) J. Am. Chem. Soc. 98:3655. Removal of the 5′ protecting group of the newly added monomer (the “deprotection” step) is typically accomplished by reaction with acidic solution to yield a free 5′ hydroxyl group, which can be coupled to the next protected nucleoside phosphoramidite. This process is repeated for each monomer added until the desired sequence is synthesized.
According to some protocols, the synthesis cycle of couple, cap, oxidize, and deprotect is shortened by omitting the capping step or by taking the oxidation step ‘outside’ of the cycle and performing a single oxidation reaction on the completed chain. For example, oligonucleotide synthesis according to H-phosphonate protocols will permit a single oxidation step at the conclusion of the synthesis cycles. However, coupling yields are less efficient than those for phosphoramidite chemistry and oxidation requires longer times and harsher reagents than amidite chemistry.
The chemical group conventionally used for the protection of nucleoside 5′-hydroxyls is dimethoxytrityl (“DMT”), which is removable with acid. Khorana (1968) Pure Appl. Chem. 17:349; Smith et al. (1962) J. Am. Chem. Soc. 84:430. This acid-labile protecting group provides a number of advantages for working with both nucleosides and oligonucleotides. For example, the DMT group can be introduced onto a nucleoside regioselectively and in high yield. Brown et al. (1979) Methods in Enzymol. 68:109. Also, the lipophilicity of the DMT group greatly increases the solubility of nucleosides in organic solvents, and the carbocation resulting from acidic deprotection gives a strong chromophore, which can be used to indirectly monitor coupling efficiency. Matteucci et al. (1980) Tetrahedron Lett. 21:719. In addition, the hydrophobicity of the group can be used to aid separation on reverse-phase HPLC. Becker et al. (1985) J. Chromatogr. 326:219.
However, use of DMT as a hydroxyl-protecting group in oligonucleotide synthesis is also problematic. The N-glycosidic linkages of oligodeoxyribonucleotides are susceptible to acid catalyzed cleavage (Kochetkov et al., Organic Chemistry of Nucleic Acids (New York: Plenum Press, 1972)), and even when the protocol is optimized, recurrent removal of the DMT group with acid during oligonucleotide synthesis results in depurination. Shaller et al. (1963) J. Am. Chem. Soc. 85:3821. The N-6-benzoyl-protected deoxyadenosine nucleotide is especially susceptible to glycosidic cleavage, resulting in a substantially reduced yield of the final oligonucleotide. Efcavitch et al. (1985) Nucleosides & Nucleotides 4:267. Attempts have been made to address the problem of acid-catalyzed depurination utilizing alternative mixtures of acids and various solvents; see, for example, Sonveaux (1986) Bioorganic Chem. 14:274. However, this approach has met with limited success. McBride et al. (1986) J. Am. Chem. Soc. 108:2040.
Conventional synthesis of oligonucleotides using DMT as a protecting group is problematic in other ways as well. For example, cleavage of the DMT group under acidic conditions gives rise to the resonance-stabilized and long-lived bis(p-anisyl)phenylmethyl carbocation. Gilham et al. (1959) J. Am. Chem. Soc. 81:4647. Protection and deprotection of hydroxyl groups with DMT are thus readily reversible reactions, resulting in side reactions during oligonucleotide synthesis and a lower yield than might otherwise be obtained. To circumvent such problems, large excesses of acid are used with DMT to achieve quantitative deprotection. As bed volume of the polymer is increased in larger scale synthesis, increasingly greater quantities of acid are required. The acid-catalyzed depurination which occurs during the synthesis of oligodeoxyribonucleotides is thus increased by the scale of synthesis. Caruthers et al, in Genetic Engineering: Principles and Methods, J. K. Setlow et al., Eds. (New York: Plenum Press, 1982).
Considerable effort has been directed to developing 5′-O-protecting groups which can be removed under non-acidic conditions. For example, Letsinger et al. (1967) J. Am. Chem. Soc. 89:7147, describe use of a hydrazine-labile benzoyl-propionyl group, and deRooij et al. (1979) Real. Track. Chain. Pays-Bas. 98:537, describe using the hydrazine-labile levulinyl ester for 5′-OH protection (see also Iwai et al. (1988) Tetrahedron Lett. 29:5383; and Iwai et al. (1988) Nucleic Acids Res. 16:9443). However, the cross-reactivity of hydrazine with pyrimidine nucleotides (as described in Baron et al. (1955) J. Chem. Soc. 2855 and in Habermann (1962) Biochem. Biophys. Acta 55:999), the poor selectivity of levulinic anhydride and hydrazine cleavage of N-acyl protecting groups (Letsinger et al. (1968), Tetrahedron Lett. 22:2621) have made these approaches impractical. Seliger et al. (1985), Nucleosides & Nucleotides 4:153, describes the 5′-O-phenyl-azophenyl carbonyl (“PAPco”) group, which is removed by a two-step procedure involving transesterification followed by β-elimination; however, unexpectedly low and non-reproducible yields resulted. Fukuda et al. (1988) Nucleic Acids Res. Symposium Ser. 19, 13, and Lehmann et al. (1989) Nucleic Acids Res. 17:2389, describe application of the 9-fluorenylmethylcarbonate (“Fmoc”) group for 5′-protection. Lehmann et al. (1989) report reasonable yields for the synthesis of oligonucleotides up to 20 nucleotides in length. The basic conditions required for complete deprotection of the Fmoc group, however, lead to problems with protecting group compatibility. Similarly, Letsinger et al. (1967), J. Am. Chem. Soc. 32:296, describe using the p-nitrophenyloxycarbonyl group for 5′-hydroxyl protection. In all of the procedures described above utilizing base-labile 5′-O-protecting groups, the requirements of high basicity and long deprotection times have severely limited their application for routine synthesis of oligonucleotides.
Still an additional drawback associated with conventional oligonucleotide synthesis using DMT as a hydroxyl-protecting group is the necessity of multiple steps, particularly the post-synthetic deprotection step in which the DMT group is removed following oxidation of the internucleoside phosphite triester linkage to a phosphorotriester. It would be desirable to have a synthesis protocol where the hydroxyl-protecting group could be removed concurrently with oxidation, such that the final two steps involved in nucleotide addition, namely oxidation and deprotection, could be combined.
The problems associated with the use of DMT are exacerbated in solid phase oligonucleotide synthesis where “microscale” parallel reactions are taking place on a very dense, packed surface. Applications in the field of genomics and high throughput screening have fueled the demand for precise chemistry in such a context. Thus, increasingly stringent demands are placed on the chemical synthesis cycle as it was originally conceived, and the problems associated with conventional methods for synthesizing oligonucleotides are rising to unacceptable levels in these expanded applications.
Our own previous research on using carbonate protecting groups resulted in the discovery that carbonate groups could be used with good effect to reduce depurination during the synthesis and to combine the oxidation step with the removal of the carbonate protecting group. U.S. Pat. No. 6,222,030 to Dellinger et al. (Apr. 24, 2001).
Oligonucleotides may be useful as diagnostic or screening tools, for example, on polynucleotide arrays. Such arrays include regions of usually different sequence polynucleotides arranged in a predetermined configuration on a substrate. These regions (sometimes referenced as “features”) are positioned at respective locations (“addresses”) on the substrate. The arrays, when exposed to a sample, will exhibit an observed binding pattern. This binding pattern can be detected upon interrogating the array. For example all polynucleotide targets (for example, DNA) in the sample can be labeled with a suitable label (such as a fluorescent compound), and the fluorescence pattern on the array accurately observed following exposure to the sample. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding pattern will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
Polynucleotide arrays can be fabricated by depositing previously obtained polynucleotides onto a substrate, or by in situ synthesis methods. The in situ fabrication methods include those described in WO 98/41531 and the references cited therein. The in situ method for fabricating a polynucleotide array typically follows, at each of the multiple different addresses at which features are to be formed, the same conventional iterative sequence used in forming polynucleotides on a support by means of known chemistry.
The foregoing methods of preparing polynucleotides are well known and described in detail, for example, in Caruthers (1985) Science 230: 281-285; Itakura et al., Ann. Rev. Biochem. 53: 323-356; Hunkapillar et al. (1984) Nature 310: 105-110; and in “Synthesis of Oligonucleotide Derivatives in Design and Targeted Reaction of Oligonucleotide Derivatives”, CRC Press, Boca Raton, Fla., pages 100 et seq.; U.S. Pat. No. 4,458,066; U.S. Pat. No. 4,500,707; U.S. Pat. No. 5,153,319; U.S. Pat. No. 5,869,643; EP 0294196, and elsewhere. The phosphoramidite and phosphite triester approaches are most broadly used, but other approaches include the phosphodiester approach, the phosphotriester approach and the H-phosphonate approach.
In the case of array fabrication, different monomers may be deposited at different addresses on the substrate during any one iteration so that the different features of the completed array will have different desired polynucleotide sequences. One or more intermediate further steps may be required in each iteration, such as the conventional oxidation and washing steps.
Each iteration of the foregoing conventional sequence can have a very high yield (over 90%), with each step being relatively rapid (requiring less than a minute). Thus, the foregoing conventional sequence is ideal for preparing a particular polynucleotide on a packed column. Whether the preparation requires four or five minutes is usually not great concern. However, when it is desired to mass produce a polynucleotide array with hundreds or more typically, thousands, of features each carrying different polynucleotides requiring ten, twenty or more cycles, the time taken for each step in each cycle at each feature becomes much more important. Furthermore, each step in the cycle requires its own solutions and appropriate system of delivery to the substrate during in situ array fabrication, which complicates an in situ array fabrication apparatus and can lead to more waste. It would be desirable then, to provide a means of fabricating an array by the in situ process with a simplified synthesis cycle requiring requiring fewer steps and/or less time to complete each cycle. It would further be desirable if the number of solutions required for each cycle could be reduced.